京都大学 / 新制・課程博士 / 博士(農学) / 甲第24911号 / 農博第2574号 / 新制||農||1102(附属図書館) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 舟場 正幸, 教授 太田 毅, 教授 横井 伯英 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Orientador: Fausto Berzin / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-15T03:24:24Z (GMT). No. of bitstreams: 1 Sakabe_FabianaForti_D.pdf: 2084817 bytes, checksum: b0340d79b05862a9990b4042a871cf3b (MD5) Previous issue date: 2010 / Resumo: Tem sido demonstrado que os sinais eletromiográficos e a histomorfometria diferem ao longo do ventre muscular e entre os gêneros, porém a relação entre esses parâmetros não está bem estabelecida. O objetivo deste trabalho foi avaliar a eletromiografia (EMG) em diferentes intensidades de contração (IC) isométrica voluntária (10 a 100%) e posicionamentos de eletrodos (PE) sobre o músculo vasto lateral proximal (VLP) e distal (VLD), assim como a histomorfometria em ambos os gêneros. Participaram 11 mulheres e 7 homens (22,0+2,1 e 23,3+2,5 anos, respectivamente), sem lesões nos membros inferiores. Inicialmente foi obtida uma linha de base para determinação da força máxima de extensão da perna (célula de carga MM-100 (KRATOS®)). Para a coleta da EMG foi utilizado o módulo de aquisição EMG-1000 da Lynx® (16 bits de resolução, filtro passa-banda de 20-1000 Hz e freqüência de amostragem 2000 Hz). Dois eletrodos de superfície ativos simples diferencial (Lynx®, ganho 20x) com distância intereletrodo de 10mm foram posicionados sobre o VLP e VLD. O eletrodo de referência foi fixado à tuberosidade da tíbia. O sinal foi coletado simultaneamente nos eletrodos e na célula de carga em diferentes IC por 5 segundos, repetida por 3 vezes e com intervalo de 1 minuto. A EMG foi processada no software Matlab® 6.5.1, sendo determinados o RMS e a freqüência mediana (FM). Após a EMG, foi realizada a biópsia do VLP e VLD com agulha de Bergström. Os cortes (12um) foram realizados em criostato (MICRON HM 505E a -25°C). Os principais tipos de fibras (TF) musculares (I, IIA e IID) foram delineados pela técnica dxa mATPase e a área das fibras (um2) foi analisada no software Image Manager (Leica Microsystems®). Foram utilizados os testes ANOVA com post-hoc de Tukey, o teste T de Student pareado e não pareado e a correlação de Pearson. Com o aumento da IC (10100% CIVM) houve elevação significativa do RMS (em ambos os PE e gêneros). Para a FM, de maneira geral, não houve variação com o aumento da IC (nos PE e gêneros). A força e o RMS bruto foram maiores nos homens em relação às mulheres para os dois eletrodos. A FM não diferiu entre os gêneros. Para a distribuição (%) dos TF, não houve variação tanto nos gêneros quanto nos PE. Entretanto, a área dos diferentes TF foi maior nos homens do que nas mulheres e de maneira geral, não houve variação entre os PE. Verificou-se correlação positiva entre força x área I e área II, RMS x área I e área II. Não houve correlação entre área das fibras e FM, nem entre força e FM. De acordo com os resultados obtidos, pode-se verificar que o percentual de fibras não variou entre os PE e gêneros, porém os homens apresentaram os TF, a força e o RMS maiores do que as mulheres. A FM não diferiu entre os gêneros. Deste modo, a área das fibras musculares influenciou o RMS e a força do músculo vasto lateral, sendo a diferença entre os gêneros provavelmente devido a essa variável. / Abstract: It has been shown that electromyography signal (EMGs) and histomorphometry are different along the muscle belly and between genders, although the relationship between these parameters is not clear. The aim of this study was to evaluate the EMGs in different intensities of volunteer isometric muscle contraction (10-100%) and electrodes positions over the belly of vastus lateralis muscle [proximal (VLP) and distal (VLD) portions], as well as the histomorphometry for both genders. Eleven women (22,0 ± 2,1 years) and seven men (23,3 ± 2,5 years), took part in the study. All subjects had no lesions in lower limbs. At first a baseline was made to determine the maximum force of leg extension [MM-100 load cell (KRATOS®)]. For EMG recording an EMG-1000 acquisition module (Lynx®) was used (16 bits of resolution, band-pass filter of 201000Hz and sampling frequency of 2000Hz). Two simple differential surface active electrodes (Lynx®, 20 times gain) separated by 10mm were placed over VLP and VLD muscles bellies. Reference electrode was attached to the anterior tibial tuberosity of the evaluated leg. EMGs was recorded simultaneously in both electrodes in different intensities of contraction for 5 seconds, repeated for 3 times and with one-minute intervals among contractions. Load cell signals were also recorded. EMGs were analyzed in Matlab® 6.5.1 software and RMS and median frequency (MF) values were obtained. After EMGs recordings, the muscle biopsies of VLP and VLD were performed using a Bergström needle. Histological sections (12um) were performed in cryostat (MICRON HM 505E at -25°C). The main types of muscle fibers (I, IIA and IID) were identified by mATPase technique and fibers' area (um2) was analyzed in Image Manager software (Leica Microsystems®). ANOVA test and Tukey post-hoc test, paired and non-paired Student t test, paired Student t test and Pearson test (correlations). A significant elevation of RMS was observed with the rise of contraction intensity (10-100% maximum voluntary isometric contraction - MVIC) for both genders and electrodes positions. In general, there were no variations in MF with the rise of contraction intensity (for both genders and electrodes positions). Force and absolute RMS values were higher in men compared to women for both electrodes. MF was not different between genders. For fiber types distribution (%) there was no difference between genders and between electrodes positions. However, male's fibers' areas were greater than women's and, as a general matter, there was no difference between the electrodes. A positive correlation was observed between force x type I and II fiber areas, RMS x type I and II fiber areas. There was no significant correlation between fibers' area and MF and between force and MF. According to our results the fiber type's distribution was not different between genders and electrodes; nevertheless, higher RMS and force values, as well as greater fibers' areas, were found in male group. MF was not different between genders. Finally, muscle fibers' area did influence RMS and force of vastus lateralis muscle and we suggest that this could be the reason of the observed differences between genders. / Doutorado / Anatomia / Doutor em Biologia Buco-Dental

Metabolic Syndrome Insulin Resistance Is Associated with Discordant Distribution of GLUT4 and the Insulin Receptor in Fast-Twitch and Slow-Twitch Muscle Fiber Types We have previously shown that We have previously shown that strength training alone improved insulin responsiveness in sedentary controls but not in metabolic syndrome subjects. Immunoblots of metabolic syndrome subjects[apos] muscle homogenates showed training-related increases in GLUT4 and mitochondrial enzymes was half that seen in the controls. To determine if this was due to changes primarily in fast-twitch fibers (strength fibers), we performed immunohistochemical (IHC) studies on muscle sections from these subjects to quantify fiber-specific changes in GLUT4, phospho-AMPK, phospho-mTOR, ATP synthase, and the insulin receptor. Signal intensity in confocal microscopic images was digitally quantified and the amount in each fiber type was adjusted by the fiber composition and the average size of each fiber type. Fiber type was classified using monoclonal antibodies against slow-twitch (type 1 fibers) and fast-twitch (type 2a and 2b fibers) myosin heavy chains. At baseline, both groups had slightly more insulin receptor in slow-twitch fibers, and most of the ATP synthase (mitochondrial marker) was in fast-twitch fibers. In controls, 55% of GLUT4 was in slow-twitch fibers, whereas metabolic syndrome subjects had only 33% of their GLUT4 in slow-twitch fibers. The IHC data showed modest increases in GLUT4 (9-25%), and substantial increases of ATP synthase (55-95%), and insulin receptors (44-104%) in both fiber types in both groups. Training-related increases were seen in phospho-AMPK (25% in slow-twitch, 15% in fast-twitch) only in the control subjects but no change in phospho-mTOR in either subject group. At baseline, metabolic syndrome subjects[apos] muscle had 56% of insulin receptors expressed in slow-twitch fibers, but only 33% of the GLUT4 was in these fibers. Thus, the untrained muscle composition of the metabolic syndrome subjects exhibited a mismatch between insulin receptors and GLUT4 in their fiber-specific distributions. This mismatch may contribute to the insulin resistance seen in the metabolic syndrome and may be involved in the diminished insulin sensitivity response to strength training in these subjects.

The fast-skeletal-muscle-fiber-specific expression of the troponin I(fast) (TnIfast) gene is driven by an Intronic Regulatory Element (IRE) located within the first intron of the gene. The IRE is a 148 bp transcriptional enhancer that contains several known and suspected cis-regulatory elements. These include the E-box, the closely-spaced MEF2 site and CACT box, the CACC site, and the CAGG element. Previous loss-of-function studies performed using the quail TnIfast IRE suggest that its activity depended on the MEF2 and CACT elements. The goal of my thesis research was to determine whether the MEF2 and CACT sites were not only necessary, but also sufficient, to support IRE activity. I prepared head-to-tail multimers of a 27-bp IRE segment that consisted largely of the near-adjacent MEF2 and CACT elements and did not contain any other known/suspected elements. These multimers were cloned upstream of a reporter gene consisting of the minimal promoter of the quail TnIfast gene linked to sequences encoding human placental alkaline phosphatase. The transcriptional capabilities of the constructs were assessed by gene transfer into the mouse soleus muscle in vivo by intramuscular injection/electroporation, and histochemical analysis of reporter enzyme plap expression including quantitative microdensitometry. I found that expression of these constructs was readily detectable and that it was markedly reduced by prior mutation of the CACT and, especially, of the MEF2 sites. These data indicate that the short DNA segment containing MEF2 and CACT elements is sufficient to drive expression in skeletal muscle and confirms the functional importance of these specific elements. / Although constructs containing the wild-type IRE 27-bp region were expressed, there was little preferential expression in fast fibers, in contrast to expression driven by the complete 148-bp IRE. Thus my results indicate that the MEF2 and CACT elements are not sufficient to drive fast fiber-type-specific expression, and suggest that additional elements outside of the 27-bp region tested are also necessary for fiber-type-specificity.

No description available.